We supported the preclinical development of the TDL clinical protocol (P.I. Michael Bishop;07-C-0064) by establishing the feasibility of expanding TDL in cultures through the use of anti CD3/anti CD8 beads, resulting in a product with high viability, donor origin and T cell content, very low residual B cell numbers, free of endotoxin or contamination;In this process, we developed methods for viably dissociating cells and culturing them under good manufacturing process (GMP) standards. Furthermore we supported the clinical approval process by developing the necessary documentation of standard operating procedures and certificates of analysis for product release, assembling GMP reagents lists, and providing links to established investigational new drug (IND) protocols for manufacture of an clinical product suitable for infusion into patients. Following the approval of this protocol, we have continued to assess T cell expansion and clearance of B cell lymphoma and Hodgkins disease populations in the TDL expansion cultures in the first six patients. We have characterized the TDL product from both preclinical test cultures and from the first clinical trial cultures, using multiparameter flow cytometry and cytokine production assays. We have demonstrated that the TDL expansion cultures result in the disappearance of lymphoma cells and a marked decline in the frequency of regulatory T cells found in the original tumor population. The final product contains more than 90% T cells, primarily T-Bet+ Th1/Tc1 cells, that have elevated expression of effector molecules including CD40L, NKG2D, and perforin, and produce primarily IFN-gamma on stimulation. These assays will form the basis for tests of efficacy and specificity of anti-tumor activity that will be used to optimize the TDL product. Furthermore we have supported a clinical initiative to make this therapy available to a broader patient population by demonstrating the feasibility of using patient bone marrow (rather than surgically excised lymphoma nodules) as a basis for generating TDL in patients with marrow-resident tumor populations. We have successfully expanded replicate cultures from patient marrow collected following allogeneic transplant, demonstrating significant expansion of donor-derived T cells that meet criteria for T cell numbers and viability, donor chimerism, removal of tumor cells and microbial standards. These tests have supported submission of a protocol amendment providing for use of marrow. Finally we have initiated development of alternative culture conditions to optimize not only the numerical expansion of donor-derived T cells from the lymphoma tissue, but also anti-tumor activity and persistence in vivo after re-infusion. We have altered the cytokine milieu of the culture to enhance retention of activated CD8 effectors.